Characterization of Biological Nanoparticle Subsets

在安装疫苗分支Astrios-EQ（2014年年中安装，与我们于2013年与Beckman-Coulter共同启动的CRADA一起）后的两年中，我们开发了用于荧光检测和校准、计数和高通量分选的nanoFACS方案，以通过nanoFACS从血浆和其他生物流体中分选EV子集。使用多个不同的囊泡群体，我们发现nanoFACS分选是唯一能够产生高保真EV子集（95 - 97%纯度），具有功能性生物货物（RNA和蛋白质）。在过去的9 - 10个月里，我们的重点一直是将这种方法应用于临床重要研究，用于研究癌症疾病负担，转移潜力和治疗反应。作为ACI计划的一部分，我们开发的NanoFACS方法是目前唯一能够基于单个EV特征而不是批量属性分析和分类功能性EV的方法。此外，我们已经设计了一类新的标签（分子纳米标签），可以与nanoFACS一起使用，不仅可以提高EV表位的检测，还可以通过流式细胞术进行单分子计数。为这个nanoFACS项目组装的专业仪器和专业知识在世界其他任何地方都不存在，是NCI的独特资产。我们现在有一个独特的机会来利用临床中心的nanoFACS计划，以证明这种方法对肿瘤学，免疫学和个性化医疗的潜在影响。为了在肿瘤源性EV研究中利用nanoFACS作为液体活检的目标，我们已经测试并确认了癌症和癌症干细胞标志物的nanoFACS分辨率：PSMA，CD147，CD44和CD26。在过去的6个月里，我们还发现免疫细胞标志物如MHC-II和PD-L1可以识别不同的EV亚群。我们预测，在MHC-II EV亚群上表达或作为货物包含的辅助受体和miRNA将与产生这些EV的细胞或生物体的免疫应答相关。我们已经验证了细胞系衍生EV上的PD-L1检测，并开始研究生物流体衍生EV上的PD-L1检测。我们正在与James Gulley和Tim Greten合作，以确定肿瘤衍生EV或抗原呈递细胞衍生EV上的循环PD-L1水平是否与PD-L1的肿瘤活检水平以及对PD-L1/PD-1导向疗法的反应相关。同样，我们现在正在与高级前列腺小组的成员合作，以确定具有PSMA，EpCAM和CD 147的肿瘤源性EV是否与前列腺癌疾病负担，预后和治疗反应相关。

In the two years since the installation of the Vaccine Branch Astrios-EQ (installed mid-2014, in conjunction with a CRADA that we initiated with Beckman-Coulter in 2013), we developed nanoFACS protocols for fluorescence detection and calibration, for counting, and for high-throughput sorting, to sort EV subsets from plasma and other biofluids by nanoFACS. Using multiple distinct vesicle populations, we find that nanoFACS sorting is uniquely able to produce high fidelity EV subsets (95-97% pure), with functional biological cargo (RNA and protein). During the past 9-10 months, our focus has been exclusively on application of this method for clinically important studies, for the study of cancer disease burden, metastatic potential, and responses to treatment. Our NanoFACS approach that we developed as part of this ACI program is the only method currently able to analyze and preparatively sort functional EVs based on single EV characteristics, rather than bulk attributes. Furthermore, we have devised a new class of labels (Molecular NanoTags) that can be used with nanoFACS, not only to improve the detection of EV epitopes, but also to enable single molecule counting by flow cytometry. The specialized instrumentation and expertise assembled for this nanoFACS program does not exist anywhere else in the world, is a distinctive asset for NCI. We now have a unique opportunity to leverage this nanoFACS program in the Clinical Center to demonstrate the potential impact of this approach for oncology, immunology, and personalized medicine. Toward the goal of utilizing nanoFACS in the study of tumor-derived EVs as a liquid biopsy, we have tested and confirmed nanoFACS resolution of cancer and cancer stem-cell markers: PSMA, CD147, CD44, and CD26. During the past 6 months, we also found that immune cell markers such as MHC-II and PD-L1 identify distinct EV subsets. We predict that the co-receptors and miRNA that are expressed on or contained as cargo of MHC-II EV subsets will correlate with immune responses- of the cells or the organism that produced those EVs. We have validated PD-L1 detection on cell line-derived EVs and are beginning to study detection of PD-L1 on biofluid-derived EVs. We are working with James Gulley and Tim Greten to determine whether circulating PD-L1 levels on tumor-derived EVs or antigen presenting cell-derived EVs correlate with tumor biopsy levels of PD-L1 and responses to PD-L1/PD-1-directed therapies. Similarly, we are working now with members of the Senior Prostate Group to determine whether tumor-derived EVs with PSMA, EpCAM, and CD147 correlate with prostate cancer disease burden, prognosis, and responses to treatment.